Nematodes are animal species that constitute a large phylum and are a type of harmful organisms parasitizing plants or animals. In general, root-knot nematodes parasitizing plants are 1 mm or shorter in length. However, they absorb nourishment from plant cell cytoplasms, and the damage caused thereby represents as much as approximately one billion dollars per year worldwide. Up to the present, approximately 70 species of root-knot nematodes belonging to the genus Meloidogyne have been identified. Since they parasitize all types of crops and a wide variety of weeds, they are reported to adversely affect over 2,000 plant species, including sweet potatoes, tomatoes, and Irish potatoes.
When a plant is infected with root-knot nematodes, no distinctive symptom that would be effective for determining parasitism at the initial stage is observed in the aerial part; however, a gall or knot begins to form below the ground. The size of such gall or knot varies depending on the species or variety thereof, and is approximately 1 to 2 mm in many cases. Thus, such gall or knot is sometimes difficult to visually observe, although egg masses laid on the surface of the gall or knot or on roots can be visually observed. The most significant symptom is a vertical crack appearing on the root or tuber, and nematodes at various developmental stages parasitize the infected root or tuber. Root-knot nematode infection not only lowers crop yields but also drastically reduces or eliminates the market value of the infected root or tuber. Also, a crack created on a root or tuber allows other pathogenic organisms to easily attack the plant, which in turn increases the likelihood of complex infection (Hooker, W. J., Compendium of Potato Diseases, pp. 97-98, 1981, The American Phytopathological Society, St. Paul Minn., U.S.A.; Jansson & Raman, Sweet Potato Pest Management, pp. 1-12, 1991, Westview Press, Boulder, Colo., U.S.A.; Jones et al., Compendium of Tomato Diseases, pp. 49-50, 1991, APS PRESS, St. Paul, Minn., U.S.A.).
Nematodes of the genus Meloidogyne parasitizing potatoes are of the following four species: Meloidogyne (M.) arenaria Chitwood; M. incognita Chitwood; M. hapla Chitwood; and M. javanica Chitwood. Among them, the Meloidogyne incognita nematode is generated with the highest frequency in potato fields worldwide (Hooker, W. J., Compendium of Potato Diseases, pp. 97-98, 1981, The American Phytopathological Society, St. Paul, Minn., U.S.A.). Nematode infection is observed in potato cultivating areas in Kyushu, Japan, where the weather is warm. Accordingly, conferment of resistance upon crops or development of integrated pest control techniques is desired.
In the case of potatoes, root-knot nematodes have been controlled for a long time via crop rotation. This technique is effective in terms of reduction of the population density of nematodes; however, control of root-knot nematodes simply via crop rotation is difficult in the case of omnivorous root-knot nematodes due to limitations concerning the cycle of crop rotation. Alternatively, the population density of root-knot nematodes can be restricted with the aid of ammonia nitrogen by adding organic fertilizers. This technique is still employed in Africa, Asia, and Central and South America at present, although it is not an ultimate method of control of root-knot nematodes. Soil fumigation with dichloropropene, methyl bromide, or the like is the best technique in terms of speed of action. This technique, however, adversely affects the ecosystem and farmers.
Currently, a technique for enhancing the nematode resistance of host potatoes has been experimentally carried out, and a variety of resistant lines have been created (Watanabe et al., Amer. Potato J. 71: 599-604, 1994; Watanabe et al., Breeding Science 45: 341-347, 1995; Watanabe et al., Breeding Science 46: 329-336, 1996; Watanabe et al. Breeding Science 49: 53-61, 1999; Watanabe & Watanabe, Plant Biotechnology 17: 1-16, 2000). Tetraploid potato cultivars that are highly resistant to nematodes, particularly to Meloidogyne incognita, have not yet been created.
Conferment of resistance using root-knot nematode-resistant diploid wild relatives upon cultivated potatoes has been attempted. Based on genetic analysis of phenotypes or breeding experiments, diploid wild relatives have been found to comprise root-knot nematode-resistance genes (Rmi), and these genes have been found to have quantitative resistance with additive effects (Iwanaga et al., J. Amer. J. Hort Sci., 114 (6): 1008-1113, 1989; Watanabe et al., Breeding Sci., 46: 323-369, 1996; Watanabe et al., Breeding Sci., 49: 53-61, 1999). In the aforementioned literature, resistance induced by such Rmi genes is reported to be unaffected by temperature and to be active at high temperatures. The Rmi is, however, not yet isolated, and the sequence thereof is not yet known. Since cultivated potatoes are autotetraploids, the heredity patterns thereof are complicated. Thus, the breeding of a useful resistant variety has not yet been realized.
At present, the positions of a group of genes resistant to the genus Meloidogyne on several gene maps have only been verified regarding tomatoes and potatoes. In the case of tomatoes, for example, the Lycopersicon. peruvianum-derived Mi gene resistant to Meloidogyne incognita Chitwood, Meloidogyne javanica Chitwood, and Meloidogyne arenaria Chitwood is reported to be located on chromosome 6 (Messeguer et al., Theor. Appl. Genet., 82: 529-536, 1991; Ho et al., Plant J., 2: 971-982, 1992). The L. peruvianum-derived Mi3 gene resistant to Meloidogyne incognita Chitwood and Meloidogyne javanica Chitwood is also reported to be located on chromosome 12 (Yaghoobi et al., Theor. Appl. Genet., 91: 457-464, 1995). The Mi gene was isolated by the group of Williamson et al., and the constitution thereof has been elucidated (Rossi et al., Proc Natl Acad. Sci, 95: 9750-9754, 1998; Milligan et al., Plant Cell, 10: 1307-1319, 1998). Since the Mi gene is affected by high temperature, the resistance thereof becomes disadvantageously inactive upon exposure to high temperatures during the initial stage of infection, i.e., 24 to 48 hours after infection.
In the case of potatoes, Rmc1 resistant to the Meloidogyne chitwoodi race 1 is reported to be located on chromosome 11 of S. bulbocastanum (Brown et al., Theor Appl. Genet., 92: 572-576, 1996). Concerning transmission of resistance to Meloidogyne incognita Chitwood, the following two possibilities have been pointed out: 1) two or more genes may be involved with resistance (Gomez et al., Amer. Potato J., 60: 353-360, 1983); and 2) cytoplasm may be involved with development of resistance (Gomez et al., Amer. Potato J., 60: 353-360, 1983; Iwanaga et al., J. Amer. Hort. Sci., 114: 1108-1013. 1989). Further, resistance to Meloidogyne incognita Chitwood is found to be additive and quantitative resistance that is controlled by 5 or 6 resistance genes (Watanabe et al., Breed. Sci., 9: 53-61, 1999; Watanabe et al. submitted).
In general, potent resistance of plants to pathogens is often very highly specific. The “gene-for-gene” hypothesis proposed by Flor (Flor, Ann. Rev. Phytopathol., 9: 275-296, 1971) describes such highly specific resistance based on the interaction between resistance genes of plants and avirulence genes of pathogens. It is generally hypothesized that a ligand-receptor model is a mechanism for gene-for-gene molecule recognition (Gabriel & Rolfe, Ann. Rev. Phytopathol. 28: 365-391, 1990).
Up to the present, the isolated resistance genes are classified into 5 groups based on functional or structural similarities of gene products (Baker et al., Science, 276: 726, 1997; Bergelson et al., Science 292: 2281-2285, 2001; Dangl and Jones, Nature 411: 826-833, 2001). The resistance genes classified as class I have nucleotide-binding sites (NBS) and leucine-rich repeats (LRR), and it is deduced that these regions are involved with signal transduction for developing resistance. Examples of the isolated genes classified as class I include: the N gene of tobacco resistant to tobacco mosaic virus (Whitham et al., Cell, 78: 1101-1105, 1994); the L6 (Lawrence et al., Plant Cell, 7: 1195-1206, 1995) and M (Anderson et al., Plant Cell, 9: 641-651, 1997) genes of flax resistant to Melampsora lini; the RPP5 (Bent, Plant Cell, 8: 1757-1771, 1996) gene of Arabidopsis thaliana resistant to Peronospora parasitica, the RPS2 (Bent et al., Science 265: 1856-1860, 1993; Mindrinos et al., Cell, 78: 1089-1099, 1994) and the RPM1 (Grant et al., Science, 269; 843-846, 1995) genes thereof resistant to Pseudomonas syringae; and the PRF (Salmeron et al., Cell 86: 123-133, 1996) gene of tomatoes resistant to Pseudomonas syringae and the I2C-1 (Ori et al., Plant Cell 9: 521-531, 1997) gene thereof resistant to Fusarium oxysporum. Further, the aforementioned L. peruvianum-derived Mi gene of tomatoes resistant to root-knot-nematodes is also found to have NBS and LRR (Milligan et al., Plant Cell 10: 1307-1319, 1998).
A protein belonging to class I has incomplete LRR on its C-terminal side and NBS on its N-terminal side. NBS is observed in ATPase, GTPase, and the like, and is constituted by 3 motifs including a P loop (Traut, Eur J. Biochem., 229: 9-19, 1994). In general, the first kinase 1a domain forms a phosphoric acid-binding loop, and the kinase 2 domain is located downstream thereof. Aspartic acid immobilized in the kinase 2 domain is deduced to adjust a metal-binding site that is necessary for migration of phosphoric acid. The kinase 3a domain located further downstream thereof has tyrosine or arginine that often interacts with purine in ATP (Traut, Eur J. Biochem., 229: 9-19, 1994). Existence of such NBS indicates that kinase activity or the G-protein plays a key role in activating resistance (Hammond-Kosack & Jones, 1997, Annu. Rev. Plant Phusiol. Plany Mol. Bioi., 48: 575-607, 1997).
The LRR domain is observed in a variety of proteins, and it is considered to be often involved with protein-protein interactions in yeast, Drosophila, human, or other species (Kobe & Deisenhofer, Nature, 366: 751-756, 1993). Concerning plant resistance to pests, however, it is deduced that the LRR domain functions as a ligand-binding domain produced from avirulence (Avr) genes or facilitates interactions between the products of resistant (R) genes and other proteins involved with defense signal transduction (Bent, Plant Cell, 8: 1757-1771, 1996).
Potatoes are major crops worldwide, and they are excellent crops that are compatible with a wide range of production systems from high-input agriculture conducted in developed countries such as the U.S.A. and Japan to low-input agriculture conducted in developing countries in Africa, Asia, and Latin America. Potatoes are extensively cultivated for applications such as feeds, industrial starch, and fermentation material as well as for food such as staple food, vegetables, or snacks worldwide (Harris, P. M. The Potato Crop, Chapman and Hall, London, 1978; International Potato Center http://www.cgiar.org.cip/2004). From the viewpoint of the amount of production and calorie supply, potatoes are one of the most important crops particularly in developing countries. In these countries where the populations are drastically increasing, an increased amount of production and productivity of potatoes will be further expected for this valuable food source in years to come.
A large amount of potatoes produced have been lost due to pests, and damage caused by root-knot nematodes has been particularly serious in extensive areas covering tropical, subtropical, and temperate regions (Hooker, W. J., Compendium of Potato Diseases, pp. 97-98, 1981, The American Phytopathological Society, St. Paul Minn., U.S.A.).
Unfortunately, there is no ultimate solution for the damage caused by root-knot nematodes, and thus, elucidation of functions and structures of resistance genes has been awaited.